Background: Autophagy is a process that cells use to degrade and recycle cellular proteins, however, the role of autophagy in kidney fibrosis remains largely unknown. Results: Autophagy is responsible for the intracellular degradation of type I collagen. Conclusion: Autophagy negatively regulates and prevents excess collagen accumulation in the kidney. Significance: Our findings implicate a novel role of autophagy as a cytoprotective mechanism against renal fibrosis.

Autophagy can lead to cell death in response to stress, but it can also act as a protective mechanism for cell survival. We show that TGF-␤1 induces autophagy and protects glomerular mesangial cells from undergoing apoptosis during serum deprivation. Serum withdrawal rapidly induced autophagy within 1 h in mouse mesangial cells (MMC) as determined by increased microtubule-associated protein 1 light chain 3 (LC3) levels and punctate distribution of the autophagic vesicle-associated-form LC3-II. We demonstrate that after 1 h there was a time-dependent decrease in LC3 levels that was accompanied by induction of apoptosis, evidenced by increases in cleaved caspase 3. However, treatment with TGF-␤1 resulted in induction of the autophagy protein LC3 while suppressing caspase 3 activation. TGF-␤1 failed to rescue MMC from serum deprivation-induced apoptosis upon knockdown of LC3 by siRNA and in MMC from LC3 null (LC3 ؊/؊ ) mice. We show that TGF-␤1 induced autophagy through TAK1 and Akt activation, and inhibition of PI3K-Akt pathway by LY294002 or dominant-negative Akt suppressed LC3 levels and enhanced caspase 3 activation. TGF-␤1 also up-regulated cyclin D1 and E protein levels while down-regulating p27, thus stimulating cell cycle progression. Bafilomycin A1, but not MG132, blocked TGF-␤1 down-regulation of p27, suggesting that p27 levels were regulated through autophagy. Taken together, our data indicate that TGF-␤1 rescues MMC from serum deprivationinduced apoptosis via induction of autophagy through activation of the Akt pathway. The autophagic process may constitute an adaptive mechanism to glomerular injury by inhibiting apoptosis and promoting mesangial cell survival.

Renal fibrosis is the hallmark of virtually all progressive kidney diseases and strongly correlates with the deterioration of kidney function. The renin-angiotensin-aldosterone system blockade is central to the current treatment of patients with chronic kidney disease (CKD) for the renoprotective effects aimed to prevent or slow progression to end-stage renal disease (ESRD). However, the incidence of CKD is still increasing, and there is a critical need for new therapeutics. Here, we review novel strategies targeting various components implicated in the fibrogenic pathway to inhibit or retard the loss of kidney function. We focus, in particular, on anti-fibrotic approaches that target transforming growth factor (TGF)-β1, a key mediator of kidney fibrosis, and exciting new data on the role of autophagy. Bone morphogenetic protein (BMP)-7 and connective tissue growth factor (CTGF) are highlighted as modulators of pro-fibrotic TGF-β activity. BMP-7 has a protective role against TGF-β1 in kidney fibrosis, whereas CTGF enhances TGF-β-mediated fibrosis. We also discuss recent advances in the development of additional strategies for anti-fibrotic therapy. These include strategies targeting chemokine pathways via CC chemokine receptor 1 and 2 to modulate the inflammatory response, inhibition of phosphodiesterase to restore nitric oxide (NO)-cyclic 3′,5′ guanosine monophosphate (cGMP) function, inhibition of NADPH oxidase 1 (Nox1) and 4 (Nox4) to suppress reactive oxygen species production, as well as inhibition of endothelin-1 or tumor necrosis factor-α to ameliorate progressive renal fibrosis. Furthermore, a brief overview of some of the biomarkers of kidney fibrosis currently being explored that may improve the ability to monitor anti-fibrotic therapies. It is hoped that evidence based on the preclinical and clinical data discussed in this review leads to novel anti-fibrotic therapies effective in patients with CKD to prevent or delay progression to ESRD.

In progressive kidney diseases, fibrosis represents the common pathway to end-stage kidney failure. Transforming growth factor-β1 (TGF-β1) is a pleiotropic cytokine that has been established as a central mediator of kidney fibrosis. Emerging evidence demonstrates a complex scheme of signaling networks that enable multifunctionality of TGF-β1 actions. Specific targeting of TGF-β signaling pathway is seemingly critical and attractive molecular therapeutic strategy. TGF-β1 signals through the interaction of type I (TβRI) and type II (TβRII) receptors to activate distinct intracellular pathways involving the Smad and the non-Smad. The Smad signaling axis is known as the canonical pathway induced by TGF-β1. Importantly, recent investigations show that TGF-β1 also induces various non-Smad signaling pathways. In this review, we focus on current insights into the mechanism and function of Smad-independent signaling pathway via TGF-β-activated kinase 1 (TAK1) and its role in mediating the profibrotic effects of TGF-β1.

During vascular injury, the proliferation and migration of smooth muscle cells leads to characteristic neointima formation, which can be exacerbated by genetic depletion of caveolin-1 or heme oxygenase 1 (HO-1), and inhibited by carbon monoxide (CO), a byproduct of heme oxygenase 1 activity. CO inhibited smooth muscle cell proliferation by activating p38 mitogen-activated protein kinase (MAPK) and p21 Waf1/Cip1 . Exposure to CO increased caveolin-1 expression in neointimal lesions of injured aorta and in vitro by activating guanylyl cyclase and p38 MAPK. p38␤ ؊/؊ fibroblasts did not induce caveolin-1 in response to CO, and exhibited a diminished basal caveolin-1 expression, which was restored by p38␤ gene transfer. p38␤ MAPK down-regulated extracellular signalregulated protein kinase 1͞2 (ERK-1͞2), which can repress caveolin-1 transcription. Genetic depletion of caveolin-1 abolished the antiproliferative effect of CO. Thus, we demonstrate that CO, by activating p38␤ MAPK, up-regulates caveolin-1, which acts as a tumor suppressor protein that mediates the growth inhibitory properties of this gas.

We have previously demonstrated that transforming growth factor-beta(1) (TGF-beta(1)) rapidly activates the mitogen-activated protein kinase kinase 3 (MKK3)-p38 MAPK signaling cascade, leading to the induction of type I collagen synthesis in mouse glomerular mesangial cells (Wang L, Ma R, Flavell RA, Choi ME. J Biol Chem 277: 47257-47262, 2002). In the present study, we investigated the functional role of upstream TGF-beta-activated kinase 1 (TAK1) and TAK1-binding protein 1 (TAB1) in the TGF-beta(1) signaling cascade. Rapid activation of endogenous TAK1 activity by TGF-beta(1) was observed in mouse mesangial cells. Transient overexpression of TAK1 with TAB1 enhanced the activation of MKK3 and p38 MAPK with or without TGF-beta(1) stimulation, whereas a dominant-negative mutant of TAK1 (TAK1DN) suppressed TGF-beta(1)-induced activation of MKK3 and p38 MAPK. Moreover, constitutive expression of TAK1DN reduced steady-state protein levels of MKK3 and p38 MAPK as well as MKK3 phosphorylation. Increased p38alpha MAPK activity by ectopic expression of either TAB1 or wild-type p38alpha MAPK resulted in enhanced TGF-beta(1)-induced type I collagen expression. In contrast, constitutive expression of TAK1DN inhibited collagen induction. Taken together, our data indicate that TAK1 and TAB1 play a pivotal role as upstream signal transducers activating the MKK3-p38 MAPK signaling cascade that leads to the induction of type I collagen expression by TGF-beta(1). In addition, our findings also suggest that TAK1 has a novel function in regulation of the steady-state protein levels of MKK3 and p38 MAPK.

Transforming growth factor-␤1 (TGF-␤1) is a multifunctional cytokine that signals through the interaction of type I (T␤RI) and type II (T␤RII) receptors to activate distinct intracellular pathways. TAK1 is a serine/threonine kinase that is rapidly activated by TGF-␤1. However, the molecular mechanism of TAK1 activation is incompletely understood. Here, we propose a mechanism whereby TAK1 is activated by TGF-␤1 in primary mouse mesangial cells. Under unstimulated conditions, endogenous TAK1 is stably associated with T␤RI. TGF-␤1 stimulation causes rapid dissociation from the receptor and induces TAK1 phosphorylation. Deletion mutant analysis indicates that the juxtamembrane region including the GS domain of T␤RI is crucial for its interaction with TAK1. Both T␤RI-mediated TAK1 phosphorylation and TGF-␤1-induced TAK1 phosphorylation do not require kinase activity of T␤RI. Moreover, T␤RI-mediated TAK1 phosphorylation correlates with the degree of its association with T␤RI and requires kinase activity of TAK1. TAB1 does not interact with TGF-␤ receptors, but TAB1 is indispensable for TGF-␤1-induced TAK1 activation. We also show that TRAF6 and TAB2 are required for the interaction of TAK1 with T␤RI and TGF-␤1-induced TAK1 activation in mouse mesangial cells. Taken together, our data indicate that TGF-␤1-induced interaction of T␤RI and T␤RII triggers dissociation of TAK1 from T␤RI, and subsequently TAK1 is phosphorylated through TAB1-mediated autophosphorylation and not by the receptor kinase activity of T␤RI.

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